PROJECT SUMMARY Mycobacterium tuberculosis (Mtb) kills more people yearly than any other infection. Mtb is successful because it impairs key functions of macrophages and dendritic cells. Mtb survives in macrophages by preventing the normal maturation of the phagosome, creating a replicative niche that resembles an early endosome. By impairing MHC class II (MHCII) antigen presentation, Mtb undermines CD4+ T cell recognition of infected macrophages. A detailed understanding of how Mtb undermines these processes is lacking. We found that the Mtb secreted proteins, EsxG and EsxH, play a critical role in both processes. EsxG and EsxH are secreted as a heterodimer (EsxG-EsxH). We identified a host target of EsxG-EsxH: hepatocyte growth factor-regulated tyrosine kinase substrate (HGS/HRS). HRS is a component of the endosomal sorting complex required for transport (ESCRT) machinery. ESCRT plays a well-described role in trafficking cell surface receptors to the lysosome for degradation. We also found that ESCRT is required for phagosome maturation and optimal antigen presentation. Therefore, by inhibiting ESCRT, EsxG-EsxH can promote Mtb survival in multiple ways. Now, our new preliminary data suggest that EsxG-EsxH also targets oculocerebrorenal syndrome of Lowe (OCRL). OCRL is an inositol 5-phosphatase with substrate specificity for phosphatidlylinositol-4,5-bisphosphate. Like HRS, OCRL is involved in endosome and phagosome function. The central hypothesis of this grant is that EsxG-EsxH impairs the function of both OCRL and HRS, thereby blocking phagosome maturation, inhibiting antigen presentation, and promoting Mtb virulence. We propose that EsxG-EsxH impairs recruitment of both OCRL and HRS to mycobacterial phagosomes. In the case of OCRL, we hypothesize that EsxG blocks the ability of OCRL to interact with its endosomal binding partner. In the case of HRS, we hypothesize that EsxG-EsxH promote HRS ubiquitination, which locks the molecule in an inactive form. We will determine whether EsxG-EsxH inhibits OCRL, define how it impairs HRS, and test the contribution of both EsxG-EsxH targets to infection in vivo. Our previous work on this project makes us uniquely qualified to carry out these studies. Our findings will provide further important mechanistic insight into Mtb's virulence strategies. We will also elucidate the importance of OCRL and ESCRT in basic macrophage biology. Revealing the fundamental basis by which Mtb sabotages host cellular functions will lead to better therapies and vaccines for Mtb.